Molecular weight markers for western blot

ABSTRACT

A method for the preparation of molecular weight standards having peroxidase activity which can be directly identified at the time of the detection of the antigen in all western blot techniques based on the use of peroxidase.

The present invention generally relates to the techniques for the separation and detection of proteins by electrophoresis and western blot. More particularly, the invention relates to a method for the preparation of molecular weight markers (standards) for use in western blot.

TECHNOLOGICAL BACKGROUND

Since the introduction of western blot technique, radioisotope-labelled antibodies have been progressively replaced by the enzyme-labelled antibodies, mainly for their easier handling and shorter detection time. At present, the most popular methods for western blot detection of proteins are based on peroxidase conjugates (antibodies, protein A, protein G, avidin, streptavidin, and the like) whose enzymatic activity is revealed by an appropriate substrate, either chromogenic or chemiluminescent.

In common laboratory procedure, the molecular weight standards used in western blot, after transfer on membrane and staining, are marked by pencil to exactly attribute the corresponding bands. This procedure is also followed when using pre-stained standards, as the colors are generally altered or attenuated at the end of the western blot procedure, and therefore some bands disappear or are hardly detectable on the membrane. Moreover, when using chemiluminescent methods, all these standards have to be marked again on the autoradiography film, to avoid uncertain determination of the detected band.

Molecular weight standards directly detectable on the membrane exist, but they require specific conditions or treatments. For example, biotin-conjugated standards are only detectable when biotin-(strept)avidin systems are used. A set of standards sharing a common epitope, detectable by a specific antibody included within the secondary antibody solution, is commercially available (Santa Cruz Biotechnology, Santa Cruz, Calif., U.S.A.). However, this method implies purchasing both markers and secondary antibodies from a unique manufacturer. No standards detectable by the peroxidase enzymatic activity directly in the membrane used for western blot are currently available.

Methods for the detection of heme-proteins, after SDS-PAGE and transfer onto a nitrocellulose filter, based on the use of peroxidase substrates, both chromogenic and chemiluminescent, have been reported (Dorward D. W., 1993, Anal. Biochem. 209:219; Vargas C. et al., 1993, Anal. Biochem. 209:323). Furthermore, electrophoresis standards obtained by polymerization of a protein that gives raise to a discrete series of homopolymers are commercially available (Sigma, U.S.A., catalogue A9392, H2757, H2507, P8906).

DISCLOSURE OF THE INVENTION

It has now been found that the peroxidase activity can be retained and, at least partially, stabilized against denaturation by conjugation of two or more proteins having peroxidase activity or of proteins having peroxidase activity with other proteins without peroxidase activity. This allowed to prepare molecular weight standards which can directly be detected on the membrane used for western blot by reaction with a suitable peroxidase substrate.

Therefore, the invention relates to a method for the preparation of molecular weight standards for use in western blot, comprising the conjugation of two or more (poly)peptides, that can be the same or different, at least one of which has peroxidase activity, by a cross-linker, and, optionally, the subsequent separation of the conjugation products so as to cover the desired molecular weight range.

“Conjugation” herein means the link of two or more (poly)peptides by means of a suitable compound (cross-linker) capable of covalently binding the two (poly)peptides. Said cross-linkers can be selected from the group consisting of all the irreversible bifunctional cross-linkers. Disuccinimidyl suberate (DSS) or its water-soluble analog disuccinimidyl glutarate (DSG) are preferably used.

“Proteins having peroxidase activity” herein preferably means the proteins or (poly)peptides having an heme group. Cytochrome C, microperoxidase, lactoperoxidase and horseradish peroxidase are non-limitative examples of proteins having peroxidase activity which can be used according to the invention.

On the other hand, “protein without peroxidase activity” can be any non-heme protein with known molecular weight having constant, reproducible migration under the different conditions used for the electrophoresis, and capable of being easily and stably conjugated with the above mentioned cross-linker. The non-heme protein is preferably selected from the group consisting of lysozyme, carbonic anhydrase, ovalbumin and serum albumin.

Conjugation can be either single, i.e. two equal or different proteins are joined, or multiple, i.e. more than two proteins may be variously joined by means of the cross-linker. Conjugation multiplicity can be adjusted by varying the conditions of the reaction between the cross-linker and the protein or mixture of different proteins, particularly the reaction time or the reagent concentration. After completion of the reaction, a mixture of products with different conjugation multiplicities will be obtained. The conjugation provides different products, the amount of each being inversely related to its molecular weight. The product mixture can be used either as such or separated into its different components. In this way, standards covering various molecular weight ranges can be prepared. In order to define more accurately the molecular weight, for each preparation a calibration may be performed by SDS-PAGE and/or western blot or with a commercial or laboratory standard consisting of proteins with known molecular weight. It should however be considered that western blot is not an analytical technique for the determination of molecular weight. Therefore, standards whose molecular weight is not precisely determined can be used, as it is commonly the case with commercial pre-stained standards, where conjugation with the chromophore molecule induces an alteration in the electrophoretic migration.

The standards, after electrophoretic separation and transfer on the western blot membrane, can be directly detected on the membrane by reaction with a chromogenic substrate or on the autoradiography film, when using chemiluminescent substrates. Examples of conventional chromogenic and chemiluminescent substrates are: 4-chloro-1-naphthol and diaminobenzidine (chromogenic); ECL (Amersham), Supersignal (Pierce) and DuoLux (Vector) (chemiluminescent). Since cross-linking can prevent the complete protein denaturation under SDS-PAGE conditions, resulting in unpredictable migration, the apparent M.W. of the labeled proteins should preferably be estimated by calibration with commercial markers.

Furthermore, in the case of conjugation products between different proteins, the protein having peroxidase activity should preferably have low molecular weight, to increase the conjugation (peroxidase/protein) ratio thus providing better detection of the standards. Microperoxidases are preferred, such as microperoxidase MP-11, a cytochrome C peptide having 11 amino acids, or microperoxidases MP-9 and MP-8, respectively nona- or octapeptide, described by Plattner et al., 1977, Histochemistry 53:223-242, or by Harbury et al., 1960. J. Biol. Chem., 235:3649-3655, or by Kraehenbuhl et al., 1974, J. Exp. Med., 139:208-223; furthermore, MP-6 and MP-17, having 6 and 17 amino acids respectively, (Spee et al, 1996, Eur. J. Biochem. 241:215-220), can be used. As a rule, any peptide derived from peroxidase proteins by proteolysis or synthesis can be used, as far as it retains its enzymatic activity.

In principle, the molecular weight standards of the invention proved to be stable after treatment in SDS-PAGE and western blot, although the peroxidase activity is better preserved under mild denaturing conditions, avoiding high-temperature.

A further aspect of the invention relates to a kit for the preparation of molecular weight standards including, in separate containers, the protein/peptide having peroxidase activity, the cross-linker and optionally the protein having no peroxidase activity, the incubation buffer and the buffer to stop the reaction. The buffers can be Tris or lysine buffer, lysine buffer being preferred in that it provides a better protein-membrane interaction.

The effectiveness of small (poly)peptides having peroxidase activity as protein markers allows their use in place of more hindering peroxidase proteins. Secondary antibodies, avidin or streptavidin usually employed in western blot, E.L.I.S.A, immunocytochemistry and immunohistochemistry, are examples of molecules that can preferably be conjugated with the microperoxidase. The resulting conjugate will have a remarkably reduced size and a higher per-molecule number of markers, thus increasing its efficiency.

Therefore, according to a further aspect, the invention relates to the use of conjugation products prepared with small size (polypeptides having peroxidase activity, preferably cytochrome C and microperoxidase, in those techniques where enzyme labeling is required.

DESCRIPTION OF THE FIGURE

-   -   lane 1: (from bottom to top) cytochrome C monomer, dimer and         trimer;     -   lane 2: as in lane 1, after separating and mixing the oligomers         to give bands of comparable intensity;     -   lane 3: lysozyme conjugated with microperoxidase (from bottom to         top) monomer, dimer, trimer;     -   lane 4: carbonic anhydrase conjugated with microperoxidase;     -   lane 5: (from bottom to top) cytochrome C monomer, dimer and         trimer as in lane 2; ovalbumin conjugated with cytochrome C;         bovine serum albumin conjugated with cytochrome C.

The following examples illustrate the invention in greater detail.

EXAMPLES Example 1 ˜13 to 40 kDa range (cytochrome C oligomers)

Material:

20 mg cytochrome C

buffer A: 10 mM NaP_(i) pH 7.4, 150 mM NaCl

buffer B: IM DL-lysine in buffer A

buffer C: 40 mM Tris-Cl pH 7.4, 300 mM NaCl

solution D: 20 mM DSS in dimethyl sulfoxide (extemporary preparation).

-   1. Dissolve cytochrome C in 0.18 ml of buffer A. -   2. Add 0.02 ml of solution D. -   3. Stir 1 h at room temperature. -   4. Add 0.02 ml of buffer B.

If a set with bands having the same intensity is desired, continue with the subsequent steps, otherwise directly skip to step 7.

-   5. Load on a Superdex 75 column (10×300 mm) or on a column with     similar characteristics, equilibrated and eluted in buffer C at a     0.5 ml/min flow rate, separately recovering the eluted peaks. -   6. Mix each peak in amounts inversely proportional to the     chromatographic peak area. -   7. Aliquot and freeze at a temperature below −20° C.

Example 2 ˜60 to 130 kDa range (cytochrome C conjugates)

Material:

1 mg cytochrome C

3.3 mg ovalbumin

10 mg bovine serum albumin

buffer A: 10 mM NaP_(i) pH 7.4, 150 mM NaCl

buffer B: 1M DL-lysine in buffer A

solution D: 20 mM DSS in dimethyl sulfoxide (extemporary preparation).

-   1) Dissolve cytochrome C in 1 ml of buffer A. -   2) Dissolve ovalbumin in 0.8 ml of buffer A and add 0.1 ml of     solution from step 1. -   3) Dissolve bovine serum albumin in 0.8 ml of buffer A and add 0.1     ml of solution from step 1. -   4) Add 0.1 ml of solution D to the solutions prepared at steps 2 and     3. -   5) Stir the solutions from step 4 for 1 h at room temperature. -   6) Add 0.1 ml of buffer B. -   7) Aliquot and freeze at temperature below −20° C.

Example 3 ˜15 to 45 kDa range (microperoxidase MP-11 conjugates)

Material:

1 mg microperoxidase MP-11

10 mg lysozyme

5 mg carbonic anhydrase

buffer A: 10 mM NaP_(i) pH 7.4, 150 mM NaCl

buffer B: 1M DL-lysine in buffer A

solution D: 20 mM DSS in dimethyl sulfoxide (extemporary preparation).

-   1. Dissolve microperoxidase in 1 ml of buffer A. -   2. Dissolve lysozyme in 0.8 ml of buffer A and add 0.1 ml of     solution from step 1. -   3. Dissolve carbonic anhydrase in 0.8 ml of buffer A and add 0.1 ml     of solution of step 1. -   4. Add 0.1 ml of solution D to the solutions prepared at steps 2 and     3. -   5. Stir the solutions from step 4 for 1 h at room temperature. -   6. Add 0.1 ml of buffer B. -   7. Aliquot and freeze at temperature below −20° C.

EXAMPLE 4 Western Blot

Duplicated samples of unconjugated cytochrome C, horseradish peroxidase, lactoperoxidase, and of conjugated proteins (with both cytochrome C and MP-11) were mixed with reducing Laemmli sample buffer: the first duplicate was heated for 5 min at 95° C., the second at 40° C., then the samples were run in parallel with Broad Molecular Weight Markers (Bio-Rad, Hercules, Calif., U.S.A.) on 15% mini-SDS-PAGE following standard procedure. After run, the gel was blotted onto 0.45 μm nitrocellulose sheets by a semi-dry cell. Membranes were washed with 20 mM TrisCl pH 7.4, 150 mM NaCl (TBS), blocked 1 h in 3% (w/v) bovine serum albumin in TBS (BT), then incubated in BT containing 0.05% (v/v) Tween-20 either 2 h or overnight to simulate different western blot procedures. After washing in TBS containing 0.25% Tween-20 and further washing in TBS, the peroxidase activity was revealed.

Cytochrome C was also treated with DSS without adding any other protein, and subjected to gel-filtration on a Superdex 75 (1×30 cm) column (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated and eluted in 40 mM Tris-Cl pH 7.4, 300 mM NaCl, at 0.5 ml/min flow: each eluted peak was recovered separately.

Cytochrome C and MP-11 conjugates were found to retain peroxidase activity at the end of the western blot procedure, as summarized in Table 1A-B. Unconjugated peroxidase proteins were found to be more sensitive to high temperature treatment, which drastically decreased their enzymatic activity: cross-linker treatment is in fact likely to stabilize the protein structure against denaturation. TABLE 1 A. Cytochrome C conjugates Cytochrome C mg/ml Approximate Protein and 100 10 0.1 protein/cytochrome source KDa Mg/ml (a) C molar ratio Cytochrome C 12.4 100 + (Sigma C.2506) 10 + Ribonuclease I A 13.7 1 − 10 (Pharmacia 27-0323-01) Ovalbumin 45. 3.3 + 10 (Sigma A-7642) Bovine serum 67. 10 + 20 albumin (Bio-Rad 500-0007) B. MP-11 microperoxide conjugates MP-11 (Sigma M-6756) 1.8 kDa Approximate 0.1 mg/ml Protein/MP-11 Protein and source KDa Mg/ml (a) Molar ratio Lysozyme 14.3 10 + 12 (Sigma L-6876) Carbonic anhydrase 29 5 + 3 (Sigma C-2273) (a) the sign + o − indicates whether after western blot procedure the peroxidase activity of the conjugate was detectable or not, respectively

When cytochrome C was treated with the cross-linker, three oligomers were generated (Figure, lanes 1 and 2) which were easily separated by gel filtration. The electrophoretic mobility suggests their identification as monomers, dimers and trimers (Table 2).

The amount of each conjugate and of the cytochrome C oligomers was adjusted so as to make the signal intensity of each band as homogeneous as possible. Mixing the three species cytochrome C, ovalbumin- and serum albumin-cytochrome C, provided a valuable series of molecular weight standards (Figure, lane 5). The conjugation of MP-11 with lysozyme produced three species of lysozyme oligomers, all of them MP-11 conjugates (Table 1B and Figure, lane 3), monomers, dimers and trimers (Table 2). Conjugation of MP-11 with carbonic anhydrase produced a single band (lane 4). TABLE 2 Calibrated molecular weights of the conjugates KDa Cytochrome C, monomer 12.9 Cytochrome C, dimer 28.4 Cytochrome C, trimer 38.6 Lysozyme-MP-11, monomer 14.8 Lysozyme-MP-11, dimer 30.7 Lysozyme-MP-11, trimer 45.5 Carbonic anhydrase-MP11 31.5 Ovalbumin-cytochrome C 58.9 Bovine serum albumin-cytochrome C 131 

1. A method per the preparation of molecular weight standards for western blot, comprising the conjugation of two or more (poly)peptides, which can be the same or different, at least one of them having peroxidase activity, by means of a cross-linker.
 2. The method as claimed in claim 1, further comprising the separation of the conjugation products according to their molecular weights.
 3. A method as claimed in claims 1-2, wherein the (poly)peptide having peroxidase activity is selected from the group consisting of heme proteins/peptides.
 4. A method as claimed in claim 3, wherein said (poly)peptide having peroxidase activity is selected from the group of cytochrome C, microperoxidase, lactoperoxidase and horseradish peroxidase.
 5. A method as claimed in claims 1-2, wherein the protein having no peroxidase activity is selected from the group of lysozyme, carbonic anhydrase, ovalbumin and serum albumin.
 6. A method as claimed in any one of the preceding claims, wherein the cross linker is selected from irreversible bifunctional cross-linkers.
 7. Molecular weight standards for western blot obtainable by the method of claims 1-6.
 8. Molecular weight standards as claimed in claim 7, selected from the group consisting of: cytochrome C conjugate, microperoxidase and lysozyme conjugate, carbonic anhydrase and microperoxidase conjugate, cytochrome C and ovalbumin conjugate, cytochrome C and serum albumin conjugate.
 9. A kit for the preparation of the molecular weight standards as claimed in claims 7 and 8, comprising, in separate containers, the (poly)peptide having peroxidase activity and the cross-linker, and optionally the protein having no peroxidase activity and the incubation buffer.
 10. The use of a (poly)peptide having peroxidase activity for the preparation of a molecular weight standard for western blot.
 11. The use as claimed in claim 10, wherein said (poly)peptide having peroxidase activity is selected from heme (poly)peptides.
 12. The use of small size (poly)peptides having peroxidase activity as protein markers in those techniques that require enzyme labeling. 